Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1999 Oct;77(4):2145–2152. doi: 10.1016/S0006-3495(99)77055-4

Proton circulation during the photocycle of sensory rhodopsin II.

J Sasaki 1, J L Spudich 1
PMCID: PMC1300495  PMID: 10512834

Abstract

Sensory rhodopsin II (SRII) in Halobacterium salinarum membranes is a phototaxis receptor that signals through its bound transducer HtrII for avoidance of blue-green light. In the present study we investigated the proton movements during the photocycle of SRII in the HtrII-free and HtrII-complexed form. We monitored sustained light-induced pH changes with a pH electrode, and laser flash-induced pH changes with the pH indicator pyranine using sealed membrane vesicles and open sheets containing the free or the complexed receptor. The results demonstrated that SRII takes up a proton in M-to-O conversion and releases it during O-decay. The uptake and release are from and to the extracellular side, and therefore SRII does not transport the proton across the membrane. The pH dependence of the SRII photocycle indicated the presence of a protonatable group (pK(a) approximately 7.5) in the extracellular proton-conducting path, which plays a role in proton uptake by the Schiff base in the M-to-O conversion. The extracellular proton circulation produced by SRII was not blocked by HtrII complexation, unlike the cytoplasmic proton conduction in SRI that was found in the same series of measurements to be blocked by its transducer, HtrI. The implications of this finding for current models of SRI and SRII signaling are discussed.

Full Text

The Full Text of this article is available as a PDF (129.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balashov S. P., Imasheva E. S., Ebrey T. G., Chen N., Menick D. R., Crouch R. K. Glutamate-194 to cysteine mutation inhibits fast light-induced proton release in bacteriorhodopsin. Biochemistry. 1997 Jul 22;36(29):8671–8676. doi: 10.1021/bi970744y. [DOI] [PubMed] [Google Scholar]
  2. Bogomolni R. A., Stoeckenius W., Szundi I., Perozo E., Olson K. D., Spudich J. L. Removal of transducer HtrI allows electrogenic proton translocation by sensory rhodopsin I. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10188–10192. doi: 10.1073/pnas.91.21.10188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown L. S., Sasaki J., Kandori H., Maeda A., Needleman R., Lanyi J. K. Glutamic acid 204 is the terminal proton release group at the extracellular surface of bacteriorhodopsin. J Biol Chem. 1995 Nov 10;270(45):27122–27126. doi: 10.1074/jbc.270.45.27122. [DOI] [PubMed] [Google Scholar]
  4. Dioumaev A. K., Richter H. T., Brown L. S., Tanio M., Tuzi S., Saito H., Kimura Y., Needleman R., Lanyi J. K. Existence of a proton transfer chain in bacteriorhodopsin: participation of Glu-194 in the release of protons to the extracellular surface. Biochemistry. 1998 Feb 24;37(8):2496–2506. doi: 10.1021/bi971842m. [DOI] [PubMed] [Google Scholar]
  5. Govindjee R., Misra S., Balashov S. P., Ebrey T. G., Crouch R. K., Menick D. R. Arginine-82 regulates the pKa of the group responsible for the light-driven proton release in bacteriorhodopsin. Biophys J. 1996 Aug;71(2):1011–1023. doi: 10.1016/S0006-3495(96)79302-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Haupts U., Bamberg E., Oesterhelt D. Different modes of proton translocation by sensory rhodopsin I. EMBO J. 1996 Apr 15;15(8):1834–1841. [PMC free article] [PubMed] [Google Scholar]
  7. Hoff W. D., Jung K. H., Spudich J. L. Molecular mechanism of photosignaling by archaeal sensory rhodopsins. Annu Rev Biophys Biomol Struct. 1997;26:223–258. doi: 10.1146/annurev.biophys.26.1.223. [DOI] [PubMed] [Google Scholar]
  8. Jung K. H., Spudich J. L. Suppressor mutation analysis of the sensory rhodopsin I-transducer complex: insights into the color-sensing mechanism. J Bacteriol. 1998 Apr;180(8):2033–2042. doi: 10.1128/jb.180.8.2033-2042.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kamikubo H., Kataoka M., Váró G., Oka T., Tokunaga F., Needleman R., Lanyi J. K. Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1386–1390. doi: 10.1073/pnas.93.4.1386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kataoka M., Kamikubo H., Tokunaga F., Brown L. S., Yamazaki Y., Maeda A., Sheves M., Needleman R., Lanyi J. K. Energy coupling in an ion pump. The reprotonation switch of bacteriorhodopsin. J Mol Biol. 1994 Nov 4;243(4):621–638. doi: 10.1016/0022-2836(94)90037-x. [DOI] [PubMed] [Google Scholar]
  11. Krebs M. P., Spudich E. N., Spudich J. L. Rapid high-yield purification and liposome reconstitution of polyhistidine-tagged sensory rhodopsin I. Protein Expr Purif. 1995 Dec;6(6):780–788. doi: 10.1006/prep.1995.0009. [DOI] [PubMed] [Google Scholar]
  12. Lanyi J. K. Bacteriorhodopsin as a model for proton pumps. Nature. 1995 Jun 8;375(6531):461–463. doi: 10.1038/375461a0. [DOI] [PubMed] [Google Scholar]
  13. Lanyi J. K. Mechanism of ion transport across membranes. Bacteriorhodopsin as a prototype for proton pumps. J Biol Chem. 1997 Dec 12;272(50):31209–31212. doi: 10.1074/jbc.272.50.31209. [DOI] [PubMed] [Google Scholar]
  14. Nagel G., Kelety B., Möckel B., Büldt G., Bamberg E. Voltage dependence of proton pumping by bacteriorhodopsin is regulated by the voltage-sensitive ratio of M1 to M2. Biophys J. 1998 Jan;74(1):403–412. doi: 10.1016/S0006-3495(98)77797-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Oesterhelt D. The structure and mechanism of the family of retinal proteins from halophilic archaea. Curr Opin Struct Biol. 1998 Aug;8(4):489–500. doi: 10.1016/s0959-440x(98)80128-0. [DOI] [PubMed] [Google Scholar]
  16. Olson K. D., Deval P., Spudich J. L. Absorption and photochemistry of sensory rhodopsin--I: pH effects. Photochem Photobiol. 1992 Dec;56(6):1181–1187. doi: 10.1111/j.1751-1097.1992.tb09743.x. [DOI] [PubMed] [Google Scholar]
  17. Olson K. D., Spudich J. L. Removal of the transducer protein from sensory rhodopsin I exposes sites of proton release and uptake during the receptor photocycle. Biophys J. 1993 Dec;65(6):2578–2585. doi: 10.1016/S0006-3495(93)81295-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Perazzona B., Spudich E. N., Spudich J. L. Deletion mapping of the sites on the HtrI transducer for sensory rhodopsin I interaction. J Bacteriol. 1996 Nov;178(22):6475–6478. doi: 10.1128/jb.178.22.6475-6478.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rath P., Spudich E., Neal D. D., Spudich J. L., Rothschild K. J. Asp76 is the Schiff base counterion and proton acceptor in the proton-translocating form of sensory rhodopsin I. Biochemistry. 1996 May 28;35(21):6690–6696. doi: 10.1021/bi9600355. [DOI] [PubMed] [Google Scholar]
  20. Sasaki J., Shichida Y., Lanyi J. K., Maeda A. Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure. J Biol Chem. 1992 Oct 15;267(29):20782–20786. [PubMed] [Google Scholar]
  21. Sasaki J., Spudich J. L. The transducer protein HtrII modulates the lifetimes of sensory rhodopsin II photointermediates. Biophys J. 1998 Nov;75(5):2435–2440. doi: 10.1016/S0006-3495(98)77687-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Spudich E. N., Zhang W., Alam M., Spudich J. L. Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base-Asp-73 interhelical salt bridge. Proc Natl Acad Sci U S A. 1997 May 13;94(10):4960–4965. doi: 10.1073/pnas.94.10.4960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Spudich J. L., Bogomolni R. A. Mechanism of colour discrimination by a bacterial sensory rhodopsin. Nature. 1984 Dec 6;312(5994):509–513. doi: 10.1038/312509a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Spudich J. L., Lanyi J. K. Shuttling between two protein conformations: the common mechanism for sensory transduction and ion transport. Curr Opin Cell Biol. 1996 Aug;8(4):452–457. doi: 10.1016/s0955-0674(96)80020-2. [DOI] [PubMed] [Google Scholar]
  25. Spudich J. L. Protein-protein interaction converts a proton pump into a sensory receptor. Cell. 1994 Dec 2;79(5):747–750. doi: 10.1016/0092-8674(94)90064-7. [DOI] [PubMed] [Google Scholar]
  26. Spudich J. L. Variations on a molecular switch: transport and sensory signalling by archaeal rhodopsins. Mol Microbiol. 1998 Jun;28(6):1051–1058. doi: 10.1046/j.1365-2958.1998.00859.x. [DOI] [PubMed] [Google Scholar]
  27. Subramaniam S., Faruqi A. R., Oesterhelt D., Henderson R. Electron diffraction studies of light-induced conformational changes in the Leu-93 --> Ala bacteriorhodopsin mutant. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1767–1772. doi: 10.1073/pnas.94.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Subramaniam S., Gerstein M., Oesterhelt D., Henderson R. Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin. EMBO J. 1993 Jan;12(1):1–8. doi: 10.1002/j.1460-2075.1993.tb05625.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Takahashi T., Yan B., Mazur P., Derguini F., Nakanishi K., Spudich J. L. Color regulation in the archaebacterial phototaxis receptor phoborhodopsin (sensory rhodopsin II). Biochemistry. 1990 Sep 11;29(36):8467–8474. doi: 10.1021/bi00488a038. [DOI] [PubMed] [Google Scholar]
  30. Vonck J. A three-dimensional difference map of the N intermediate in the bacteriorhodopsin photocycle: part of the F helix tilts in the M to N transition. Biochemistry. 1996 May 7;35(18):5870–5878. doi: 10.1021/bi952663c. [DOI] [PubMed] [Google Scholar]
  31. Yan B., Takahashi T., Johnson R., Spudich J. L. Identification of signaling states of a sensory receptor by modulation of lifetimes of stimulus-induced conformations: the case of sensory rhodopsin II. Biochemistry. 1991 Nov 5;30(44):10686–10692. doi: 10.1021/bi00108a012. [DOI] [PubMed] [Google Scholar]
  32. Yao V. J., Spudich J. L. Primary structure of an archaebacterial transducer, a methyl-accepting protein associated with sensory rhodopsin I. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11915–11919. doi: 10.1073/pnas.89.24.11915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zhang W., Brooun A., Mueller M. M., Alam M. The primary structures of the Archaeon Halobacterium salinarium blue light receptor sensory rhodopsin II and its transducer, a methyl-accepting protein. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8230–8235. doi: 10.1073/pnas.93.16.8230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zhang X. N., Spudich J. L. HtrI is a dimer whose interface is sensitive to receptor photoactivation and His-166 replacements in sensory rhodopsin I. J Biol Chem. 1998 Jul 31;273(31):19722–19728. doi: 10.1074/jbc.273.31.19722. [DOI] [PubMed] [Google Scholar]
  35. Zhang X. N., Zhu J., Spudich J. L. The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices. Proc Natl Acad Sci U S A. 1999 Feb 2;96(3):857–862. doi: 10.1073/pnas.96.3.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhu J., Spudich E. N., Alam M., Spudich J. L. Effects of substitutions D73E, D73N, D103N and V106M on signaling and pH titration of sensory rhodopsin II. Photochem Photobiol. 1997 Dec;66(6):788–791. doi: 10.1111/j.1751-1097.1997.tb03225.x. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES